Compositions and methods for inhibiting phenyl triazole mll1-wdr5 protein-protein interaction

ABSTRACT

The present disclosure relates to the field of medicinal chemistry, in particular to a phenyl triazole MLL1-WDR5 protein-protein interaction inhibitor (I) and a preparation method thereof, and pharmacodynamics experiments prove that the compound of the present disclosure has relatively strong MLL1-WDR5 protein-protein interaction inhibition activity.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No.17/078,052, filed Oct. 22, 2020, which is a continuation ofPCT/CN2018123500, filed Dec. 25, 2018, which claims priority toCN201810365880, filed Apr. 23, 2018. The disclosures of which are hereinincorporated by reference in their entireties.

BACKGROUND

Methylation of histones plays key roles in many biological processes andis the main content of study in epigenetic regulation field.Translocation and re-arrangement of the methyl transferase MLL1 gene forhistone H3K4 can lead to mixed lineage leukemia (MLL1, acute myeloidleukemia and acute lymphoid leukemia). About 10% of leukemia patientshave MLL1 gene rearrangement. After MLL1 gene arrangement, it fuses withother chaperone proteins to form fusion proteins, and the carcinogenicMLL fusion protein is expressed. The fusion protein can interact withRNA polymerase II (Pol II) related elongation factors to form the superelongation complex (SEC). The complex can lead to abnormal expression ofthe MLL1 regulated Hox gene, which causes series of serious consequencesto induce MLL leukemia onset.

Rearrangement of the MLL1 gene occur on one allele and the wildtype MLL1is still present. When the wildtype MLL1 allele is knocked out, the MLLfusion protein alone will not lead to leukemia, and the enzyme activityof the wildtype MLL1 is necessary for the MLL1 fusion protein to causeleukemia. Thus, specific inhibition of the wildtype MLL1 enzyme activitywill cure leukemia.

Catalytic activity on H3K4 methylation by MLL1 alone is very weak andcan only result in monomethylation; the catalytic activity improvesgreatly upon the formation of the MLL1 core catalytic complex,especially the catalytic activity on H3K4me2. The WIN motif on theC-terminus of the MLL binds to WDR5, RbBP5, Ash2L and DPY30 to formcomplexes. MLL1 interacts with WDR5 directly through the C-terminus WINmotif, to mediate the interaction between the catalytic domain ofMLL1SET and other protein complexes. When WDR5 is knocked out, the levelof H3K4me2/3 decreases and the Hox gene expression level decreases.

Thus, the use of small molecule to interfere the protein-proteininteraction of MLL1-WDR5 is an effective method to inhibit MLL1enzymatic activity and lower Hox and Meis-1 gene expression levels andto block the progression of leukemia.

DETAILED DESCRIPTION

The present disclosure is in the medicinal chemistry field, morespecifically, is for a class of phenyl triazole MLL1-WDR5protein-protein interaction inhibitor, its preparation andpharmaceutical uses. An aspect of the present disclosure comprises asmall molecule compound that regulates MLL1-WDR5 protein-proteininteractions, through interference of which, it inhibits the enzymaticactivities of catalysis, down-regulates the methylation levels of H3K4,and gene expression levels of Hox and Meis-1 genes, which in turn toinduce the apoptosis of leukemia cells for the applications of leukemiatreatment. The structure of the compound in this disclosure is:

In some embodiments, X is hydrogen, methyl, methoxy or halogen groups;

In some embodiments, Y is —CH₂—, —O—, —S—, —CO—, —CH2O—, —NR5-, —CONR6-or —NR7CO—, wherein, R5, R6 and R7 are independently hydrogen, C1-C4alkyl, C1-C4 substituted

-   -   alkyl, phenyl or substituted phenyl, substituted groups are        halogen, C1-C4 alkyl, C1-C4 alkoxy, amino, hydroxy, decyl,        carboxy, cyano, trifluoromethyl or imidazolyl groups;

In some embodiments, m is 0-6;

In some embodiments, R1 is hydrogen, amino, hydroxy, decyl, carboxy,cyano, —CONH2, C1-C4 alkyl, C1-C4 alkoxy, phenyl, substituted phenyl,substituted or unsubstituted nitrogen containing or oxygen containing3-7 membered heterocyclic, —NR8COR9, —CONR10R11 or —NR10R11 groups,wherein R8 is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, phenyl orsubstituted phenyl, R9 is amino, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy,phenyl or substituted phenyl, substituted or unsubstituted nitrogencontaining or oxygen containing 3-7 membered heterocyclic, R10 and R11independently represent hydrogen, C1-C4 alkyl, phenyl or substitutedphenyl, substituted or unsubstituted nitrogen containing or oxygencontaining 3-7 membered heterocyclic groups or nitrogen or oxygencontaining 3-7 membered heterocyclic ring formed by linking R10 and R11,the said substitution groups are halogen, C1-C4 alkyl, C1-C4 alkoxy,amino, hydroxy, decyl, carboxy, cyano, trifluoromethyl or imidazolylgroups;

In some embodiments, R2 represents disubstituted or trisubstitutedhalogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitro or cyano;

In some embodiments, R3 represents amino, methylamino, aminomethyl,hydroxy, hydroxymethyl, decyl or —CONH2;

In some embodiments, R4 represents N-methylpiperazine, 1,2-dimethylpiperazine or N-methylhomopiperazine.

In some embodiments, X is preferentially hydrogen, fluorine, chlorine oralkyl groups.

In some embodiments, Y is preferentially —NR5-, CONR6- or —NR7CO—; R5,R6 and R7 are independently hydrogen, alkyl, ethyl, propyl, cyclopropylor isopropyl groups.

In some embodiments, Y is also preferentially —NR5-, —CONR6- or —NR7CO—;R5, R6 and R7 are independently substituted phenyl group, wherein thesubstitutions are methyl, ethyl, isopropyl, tert-butyl, cyclopropyl,methoxy, cyano, halogen, trifluoromethyl or imidazolyl.

In some embodiments, said substituted or unsubstituted nitrogencontaining or oxygen containing 3-7 membered heterocyclic groups arepreferentially azacyclopropyl, azacycline, tetrahydropyrrole,piperidine, cycloheximide, lactam, tetrahydrofuran, tetrahydropyran,morpholine, 1,4-oxazinidine, hexahydropyridazine, imidazoline,imidazolium, piperazine, the substituent is halogen, methyl, ethyl,phenyl, hydroxy, amino, hydroxymethyl or aminomethyl.

In some embodiments, R1 preferentially represents —NR8COR9, —CONR1OR11or —NR1OR11, while R8, R9, R10 and R11 represents C1-C4 alkyl groups.

In some embodiments, R2 preferentially represents trisubstitutions,where the substitutions are fluorine, chlorine, bromine, methyl,methoxy, nitro, trifluoromethyl or cyano groups.

The present disclosure also includes pharmaceutically acceptable saltsof compound (I) and its sovates, which all have the same pharmacologicalfunctions as that of compound (I).

Another aspect of the present disclosure comprises a drug combination,which includes compound (I) or its pharmaceutically usable salts, orsolvates, and one or more pharmaceutically usable carriers, diluents andexcipients.

Another aspect of the present disclosure comprises applications of usingcompound (I) or its pharmaceutically usable salts or solvates to preparedrugs to treat diseases mediated by the enzyme through inhibitingMLL1-WDR5 protein-protein interaction, the said diseases are such as MLLgene fusion type leukemia that can be treated through inhibition of MLL1enzymatic activities.

Dosage of the compound in this disclosure used clinically comprises 0.01mg-1000 mg/day, which can deviate from the range according to severityof diseases or different formulation.

In some of the preferred applications, compounds based on compound (I)can contain sufficient basic groups to form salts. Representative saltsinclude inorganic acid salts, organic acid salts, hydrobromide andsulfuric acid salts; pharmaceutical use organic salts, includingacetate, trifluoroacetate, lactate, succinate, fumarate, maleate,citrate, methanesulfonate, p-benzoate and p-toluenesulfonate salts.

In the meantime, another aspect of the present disclosure comprisespreparation methods of compounds related to compound (I), including thefollowing steps:

In some embodiments, R1, R2, R3, R4, X, Y and n are as definedpreviously;

In some embodiments, the intermediate Ia can be obtained through thefollowing synthesis route,

The following are some of the pharmacodynamic tests and results of thecompound in the present disclosure:

MLL1 enzymatic activities are determined by MLL1 and WDR5protein-protein interactions; MLL1 enzymatic activities affect theacetylation levels of H3K4. The H3K4 acetylation levels increaseabnormally in MLL fusion type leukemia, while the downstream Hox andMeis-1 gene expression levels increase abnormally. When MLL1-WDR5protein-protein interactions are inhibited, MLL1 catalytic activitiesdecrease, H3K4 acetylation levels decrease, Hox and Meis-1 expressionlevels decrease, (which) inhibit leukemia cell proliferation.

Biphenyl compound DDO-2084 was already reported to be able to inhibitMLL1-WDR5 protein-protein interactions, lower MLL1 catalytic activities,downregulate small molecule inhibitor expressions from Hox and Meis-1genes (Eur. J. Med. Chem. 2016, 124, 480-489.), wherein DDO-2084 was thecompound used as positive control.

TABLE 1 Inhibition activities of the compounds in this presentdisclosure over MLL1-WDR5 protein-protein interactions and relatedbiological activities MLL1- Compound WDR5 PPI Inhibition onDownregulation numbering in inhibition H3K4 of Hox and applicationactivity acetylation Meis-1 Examples^(a) (nM) level expression 1 <80 YesYes 2 <50 Yes Yes 3 <10 Yes Yes 4 <10 Yes Yes 5 <10 Yes Yes 6 <10 YesYes 7 <10 Yes Yes 8 <10 Yes Yes 9 <50 Yes Yes 10 <10 Yes Yes 12 <80 YesYes 13 <80 Yes Yes 14 <10 Yes Yes 15 <10 Yes Yes 16 <10 Yes Yes 17 <50Yes Yes 18 <50 Yes Yes 19 <50 Yes Yes 21 <80 Yes Yes 22 <10 Yes Yes 23<50 Yes Yes 24 <50 Yes Yes 25 <50 Yes Yes DDO-2084^(b) 88.7 ± 4.9 YesYes ^(a)Refer to application Examples for specific compound structures;^(b)Structure of DDO-2084:

As shown in Table 1, the compounds in this present disclosure haverelatively strong inhibitory activities on MLL1-WDR5 protein-proteininteractions.

In the meantime, RT-PCR experiments at cellular level were conductedwith some of the compounds in the present disclosure, with resultslisted in Table 1 on whether some of the compounds inhibited downstreamHox and Meis-1 gene expression levels. Results showed that all compoundsin the present disclosure with inhibitory activities on MLL1-WDR5protein-protein interactions can all inhibit downstream Hox and Meis-1gene expressions. Inhibition results at the cellular levels of some ofthe compounds on the downstream Hox and Meis-1 expression levels wereplotted in FIG. 1 , wherein FIG. 1 showed, the inhibition level of thecompound in application Example 7 at 2.5 μM reached the same level asthat of the positive control DDO-2084 at 5 μM, while the effects of thatfrom the application Example 7 were better than that of DDO-2084 at thesame 5 μM.

In addition, Western-blot experiments at cellular level were conductedwith some of the compounds in the present disclosure, with resultslisted in Table 1 on whether some of the compounds in the presentdisclosure inhibited H3K4 acetylation levels.

Results showed that all compounds in the present disclosure withinhibitory activities on MLL1-WDR5 protein-protein interactions alldownregulated H3K4 acetylation levels. The inhibition on MLL1 catalyticactivities at the cellular level for some of the compounds was plottedin FIG. 2 . As FIG. 2 shows, application Example 7 can inhibit MLL1catalytic activities in a dose-dependent manner to reduce the expressionlevels of H3K4me1/2/3, and it is showed that the results fromapplication Example 7 was better than that of DDO-2084 at the same 10 μMconcentration.

In addition, published paper (Eur. J. Med. Chem. 2016, 124, 480-489.Referred to as “Paper 1” below) has reported series of biphenylinhibitors on MLL1-WDR5 protein-protein interactions, with structuresof:

while the compounds in this present disclosure differ from them bychanging the structures on how the two benzene rings are linked, whereinposition 5 is linked by triazole groups, such changes increased thewater solubility significantly of the compounds in this presentdisclosure, reduced the toxicity of the compounds but retained theoriginal MLL1-WDR5 inhibitory activities. We have chosen some of thebiphenyl compounds from Paper 1 and some of the un-reported biphenylcompounds for solubility and toxicity tests using the same testingmethods in this present disclosure, and the results are below:

TABLE 2 Comparison of target activity and solubility of some of thecompounds in this present disclosure (Examples) and some of thecompounds in Paper 1 Structures of some compounds from MLL1- MLL1-publication (Eur. J. WDR5 PP1 Compound number and WDR5 PP1 SolubilityMed. inhibition Solubility structure of this present inhibition (pH =7.4) Chem. 2016, activity (PH = 7.4) disclosure activity (nM) μg/mL 124,480-489.) (nM) μg/mL

67.8 20

70.0 1.9

33.9 170

73.0 21

11.2 200

55.6 27

65.7 30

47.9 3.1

90.3 110

88.7 9.5

6.0 630

6.5 54

12.3 1235

8.5 125

6.7 1303

7.6 110

It is noted from the comparison of data in Table 2 for the compoundsthat, when the other groups remained the same, replacement of thebenzene rings by triazole groups in this present disclosure retainedtargeting activity and improve water solubility significantly.

In the meantime, subacute toxicity experiments were conducted toevaluate the safety of some of the compounds in the present disclosurein mouse. Some of the triazole compounds from this present disclosure(application Examples 4, 6, 7, 16 and 22) and compounds DDO-2113 andDDO-2117 from Paper 1 were given by intraperitoneal injection at 80mg/kg to female balb/c mice, 6 mice per group, for 10 consecutive daysto observe mouse survival and average body weight changes. As shown inFIG. 3 , there were no death after dosing for 10 days for some of thecompounds from this present disclosure (application Examples 4, 6, 7, 16and 22) with slight body weight increase, while after dosing with thecompounds DDO-2113 and DDO-2117 from the paper (Eur. J. Med. Chem. 2016,124, 480-489.), all mice died after dosing for 5 days with DDO-2113 at80 mg/kg and those given DOO-2117 had apparent body weight decrease. Thecomparison of post dosing survival and average body weight changes, thatno death after dosing for 10 days with the triazole compounds in thispresent disclosure with slight body weight increase in contrast to thatwith deaths and body weight decreases after dosing with diphenyl seriescompounds of DDO-2113 and DDO-2117, indicates the very good dosingsafety of the phenyltriazoles in this present disclosure.

Wherein, DDO-2113 and DDO-2117 are compounds from Paper 1 with thestructures:

Anti-proliferation experiments were conducted with leukemia cells withsome of the compounds in the present disclosure. Table 3 listed theresults of evaluation of the anti-proliferation activities for some ofthe compounds from this present disclosure conducted with acute leukemiacells, wherein MV4-11 is human acute leukemia monocyte, Molm-13 is humanacute myeloid leukemia cells, and K562 is human chronic myeloid leukemiacells. Table 3 indicated that the compounds in this present disclosurehave very good inhibition activity of different kinds of leukemia cells.

TABLE 3 Anti-proliferation activities of some of the compounds in thispresent disclosure on leukemia cells Compound numbers in the applicationGI₅₀/μM GI₅₀/μM GI₅₀/μM Examples^(a) (MV-411) (Molm-13) (K562) 1  8.9 ±0.3 11.8 ± 0.9 ND  3 44.6 ± 1.3 22.8 ± 5.5 >100 4 10.5 ± 2.0 ND^(b) 34.4± 0.9 5 15.1 ± 1.2 13.0 ± 0.6 27.5 ± 2.8 6 10.9 ± 0.2  8.2 ± 0.6 10.4 ±0.7 7  8.0 ± 1.2  9.9 ± 1.9 12.9 ± 0.4 8 17.8 ± 3.6 11.9 ± 1.5 34.1 ±1.2 9 27.4 ± 2.1 ND^(b) 35.8 ± 2.7 10 13.5 ± 1.2 13.5 ± 1.2 13.5 ± 1.211 13.5 ± 1.5 13.5 ± 1.5 13.5 ± 1.5 12 23.2 ± 3.2 ND^(b) ND^(b) 13 10.1± 1.3  9.2 ± 1.4 10.5 ± 2.0 14  9.5 ± 1.0 11.0 ± 2.0 14.1 ± 1.3 15 14.3± 1.7 18.5 ± 1.9 12.9 ± 0.2 16 15.1 ± 0.9 ND^(b) 15.0 ± 0.8 17 10.4 ±1.1  7.3 ± 0.8 21.3 ± 2.4 18 11.2 ± 1.3 15.4 ± 2.2 ND^(b) 19 13.7 ± 1.416.8 ± 2.0 13.5 ± 1.2 20  8.6 ± 0.6 10.1 ± 1.3 10.5 ± 2.0 21 12.7 ± 0.8 7.7 ± 0.9 10.5 ± 0.8 22  9.5 ± 0.4 11.2 ± 0.8 10.9 ± 0.7 23 ND^(b) 17.9± 1.6 27.7 ± 0.3 24 11.3 ± 0.7 12.9 ± 0.9 13.5 ± 2.2 25 10.9 ± 0.9  8.5± 0.7 11.3 ± 0.7 DDO-2084 17.7 ± 2.3 ND^(b) 50.5 ± 5.5 ^(a)Refer to theapplication Examples for the chemical structures; ^(b)ND indicates nottested;

The phenyl triazole compounds in this have relatively strong inhibitionactivity on MLL1-WDR5 protein-protein interactions, lowering the MLL1catalytic activities of MLL1 at the cellular level, down regulating thegene expression levels of Hox and Meis-1 and induce apoptosis ofleukemia cells, and that the phenyl triazole compounds in this presentdisclosure have shown very good water solubility and pharmaceuticalsafety, and can be used for treating leukemia.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is the RT-PCR experiment for application Example 7 to show thelowered Hoxa9 and Meis-1 gene expressions in cells

FIG. 2 is the Western Blot experiment for application Example 7 to showthe effects on MLL1 enzymatic activity in cells.

FIG. 3 is the toxicity comparison of the compounds in this presentdisclosure to some of the compounds on Article 1

EXAMPLE 1

1-(3-(5-amino-2-chloro-4-fluoro-3-methyl benzoylamino)-4-(4-methylpiperazine-1-group)phenyl)-1H-1,2,3-methyl triazole-4-carboxylatePreparation of 4-(4-methylpiperazin-1-group)-3-nitroaniline (IIb)

Dissolve 4-fluoro-3-nitroaniline (II) (6 g, 38.4 mmol) in 50 mLacetonitrile, add N-methylpiperazin (5.8 g, 6.3 mL, 57.6 mmol) and N,N-diisopropylethylamine (9.5 mL, 57.6 mmol), and heat and reflux for 12h. The crude is obtained after spin dry and purified by silica gelcolumn chromatography (dichloromethane:methanol=20:1) to obtainred-brown solid (8.9 g, 97.8%). ¹H NMR (300 MHz, DMSO-d₆) δ7.06 (d,J=8.6 Hz, 1H), 6.76 (s, 1H), 6.69 (d, J=8.5 Hz, 1H), 5.34 (s, 2H), 2.70(t, J=4.4 Hz, 4H), 2.27 (br s, 4H), 2.09 (s, 3H). m/z (EI-MS);259.1[M+Na]⁺.

Preparation of 1-(4-azido-2-nitrophenyl)-4-methylpiperazine (Ia)

Dissolve 4-(4-methylpiperazin-1-group)-3-nitroaniline (IIb)(4.0 g, 17.0mmol) in 100 mL 2M/HCl, reduce the temperature to 0° C., add 10 mL ofsodium nitrite (1.76 g, 25.5 mmol) water solution dropwise, stir for 30min at 0° C., add 10 mL sodium azide (2.2 g, 34.0 mmol) water solutiondropwise, stir 30 min at 0° C. and then stir 2 h at room temperature.The product is precipitated with 2M/NaOH at pH=9-10, vacuum filter andheat dry to obtain red-brown solid (4.0 g, 91.3%). ¹H NMR (300 MHz,DMSO-d₆) δ7.48 (d, J=2.2 Hz, 1H), 7.34-7.20 (m, 2H), 2.85 (t, J=4.7 Hz,4H), 2.31 (t, J=4.8 Hz, 4H), 2.11 (s, 3H). m/z (EI-MS): 261.1 [M+H]⁻.

Preparation of1-(4-(4-methylpiperazin-1-group)-3-nitrophenyl)-1H-1,2,3-triazole methylcarboxylate (Ib)

Dissolve 1-(4-azido-nitrophenyl)-4-methylpiperazine (Ia) (1.0 g, 3.8mmol) in 50 mL methanol, add methyl propiolate (0.96 g, 11.4 mmol),cuprous iodide (0.07 g, 0.38 mmol), N,N-diisopropylethylamine (0.12 mL,0.76 mmol), heat reflux for 48 h, filter, concentrate, and beat up withethyl acetate to obtain red-brown solid (0.8 g, 61.5%). ¹H NMR (300 MHz,DMSO-d₆) δ9.45 (s, 1H), 8.36 (d, J=2.7 Hz, 1H), 8.11-8.01 (m, 1H), 7.42(d, J=9.1 Hz, 1H), 3.80 (s, 3H), 2.99 (t, J−5.4 Hz, 4H), 2.35 (t, J=5.2Hz, 4H), 2.13 (s, 3H). m/z (EI-MS); 369.2[M+Na]⁺.

Preparation of1-(3-amino-4-(4-methylpiperazin-1-group)phenyl-1H-1,2,3-methyltriazole-4-methyl carboxylate (Ic)

Dissolve 1-(4-(4-methylpiperazin-1-group)-3-nitrophenyl)-1H-1,2,3-methyltriazole-4-methyl carboxylate (Ib) (3.8 g, 12.0 mmol) in 50 mL methanol,add Pd/C of catalytic amount, pump in hydrogen, stir at room temperaturefor 7 h, concentrate by vacuum filtering to obtain pink solid (3.0 g,78.9%). ¹H NMR (300 MHz, DMSO-d₆) δ9.28 (s, 1H), 7.28 (d, J=1.9 Hz, 1H),7.07 (d, J=1.9 Hz, 2H), 5.15 (s, 2H), 3.90 (s, 3H),

2.87 (t, J=4.5 Hz, 4H), 2.53 (br s, 4H), 2.26 (s, 3H). m/z (ESI-MS):317.1763 [M+H]⁺. Preparation of1-(3-(5-Amino-2-chloro-4-fluoro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,Methyl 2,3-triazole-4-carboxylate (1)

Dissolve1-(3-amino-4-(4-methylpiperazin-1-group)phenyl-1H-1,2,3-triazole-4-methylcarboxylate (Ic) (1.7 g, 5.3 mmol) in 100 mL anhydrous dichloromethane,add pyridine (0.43 mL, 5.3 mmol), add 20 mL dichloromethane solution of2-chloro-3-methyl-4-fluoro-5-nitrobenzoyl chloride (1.6 g, 6.4 mmol)drop wise in ice-water bath, stir 2 h at room temperature, filter undervacuum and heat dry to obtain light yellow solid; dissolve the lightyellow (2.6 g, 4.9 mmol) in ethyl acetate, add stannous chloride (5.5 g,24.4 mmol), heat and reflux for 3 h before cooling down to roomtemperature, dilute with 100 mL ethyl acetate, neutralize with saturatedsodium bicarbonate till no additional white gel-like precipitating out,filter under vacuum, wash the filter cake with ethyl acetate till noultraviolet absorption, extract the filtrate with ethyl acetate till noultraviolet absorption, combine the organic phases, dry with anhydroussodium sulfate, concentrate to obtain the crude product, beat up withethyl acetate, filter under vacuum to obtain gray-white solid 1 (2.3 g,93.9%). ¹H NMR (300 MHz, DMSO-d₆) δ9.52-9.45 (m, 2H), 8.69 (s, 1H), 7.73(dd, J=8.7, 2.7 Hz, 1H), 7.46 (d, J=8.6 Hz, 1H), 6.92 (d, J=9.2 Hz, 1H),5.53 (s, 2H), 3.92 (s, 3H), 3.00-2.90 (m, 4H), 2.51 (br s, 4H), 2.28 (d,J=2.6 Hz, 3H), 2.24 (s, 3H). (EI-MS): 502.9[M+H]⁺.

EXAMPLE 2

Preparation of1-(3-(5-Amino-2-chloro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,2,3-Triazole-4-carboxylicacid (2)

Dissolve1-(3-(5-Amino-2-chloro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-yl)phenyl)-1H-1,2,3-triazole-4-Methylformate (1) (2.3 g, 4.6 mmol) in THF, add lithium hydroxide solution(1M, 15 mL), stir for 8 h at room temperature, rotate dry to remove THFfollowed by acidification using 2M chloric acid to obtain white solid(1.7 g, 80.4%). ¹H NMR (300 MHz, DMSO-d₆) δ9.52-9.45 (m, 2H), 8.69 (s,1H), 7.73 (dd, J=8.7, 2.7 Hz, 1H), 7.46 (d, J=8.6 Hz, 1H), 6.92 (d,J=9.2 Hz, 1H), 5.53 (s, 2H), 3.92 (s, 3H), 3.00-2.90 (m, 4H), 2.50 (brs, 4H), 2.28 (d, J=2.6 Hz, 3H), 2.24 (s, 3H). (EI-MS): 488.9[M+H]⁺.

EXAMPLE 3

Preparation of1-(3-(5-Amino-2-chloro-4-fluoro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-N,N-dimethyl-1H-1,2,3 triazole-4-carboxamide (3)

Dissolve1-(3-(5-Amino-2-chloro-4-fluoro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,2,3-Triazole-4-carboxylicacid (2) (0.18 g, 0.36 mmol) in 10 mL DMF, add BOP (0.32 g, 0.72 mmol),trimethylamine (0.10 mL, 0.72 mmol) and dimethylamino hydrochloride(58.7 mg, 0.72 mmol), stir for 4 h at room temperature. Dilute thereaction mixture with 50 mL ethyl acetate, remove DMF with saturatedsodium chloride, dry the organic phase with anhydrous sodium sulphate,dry out the organic solvent with rotations to obtain raw product,isolate and purify with silica gel column chromatography(Dichloromethane:methanol=50:1) to obtain gray white solid. The yield is78.4%. ¹H NMR (300 MHz, DMSO-d₆) δ9.59 (s, 1H), 9.18 (s, 1H), 8.63 (d,J=2.5 Hz, 1H), 7.71 (dd, J=8.6, 2.6 Hz, 1H), 7.42 (d, J=8.6 Hz, 1H),6.83 (d, J=9.2 Hz, 1H), 5.50 (s, 2H), 3.11 (br s, 10H), 3.02 (s, 3H),2.70-2.69 (m, 4H), 2.24 (d, J=2.5 Hz, 3H). (EI-MS): 515.9[M+H]⁺.

EXAMPLE 4

Preparation of5-amino-2-chloro-4-fluoro-3-methyl-N-(2-(4-methylpiperazin-1-group)-5-(4-((morpholine-4-carbonyl)-1H-1,2,3-triazol-1-yl)phenyl)benzamide(4)

Using the methods in application Example 3, replace dimethylaminohydrochloride by morpholine to obtain gray white solid. The yield is67.5%. ¹H NMR (300 MHz, DMSO-d₆) δ9.61 (s, 1H), 9.24 (s, 1H), 8.64 (d,J=2.5 Hz, 1H), 7.73 (dd, J=8.6, 2.6 Hz, 1H), 7.44 (d, J=8.6 Hz, 1H),6.86 (d, J=9.2 Hz, 1H), 5.52 (s, 2H), 4.06 (s, 2H), 3.68 (s, 6H), 3.12(br s, 8H), 2.69 (s, 3H), 2.26 (d, J=2.6 Hz, 3H). (EI-MS): 558.9[M+H]⁺.

EXAMPLE 5

Preparation of1-(3-(5-Amino-2-chloro-4-fluoro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-N-(tetrahydro-2H-pyran-4-yl)-1H-1,2,3-triazole-4-carboxamide

Using the methods in application Example 3, replace dimethylaminohydrochloride with 4-aminotetrahydropyran to obtain gray white solid.The yield is 82.9%. ¹H NMR (300 MHz, DMSO-d₆) δ9.52 (s, 1H), 9.26-9.24(m, 1H), 8.65 (s, 2H), 7.71 (d, J=8.7 Hz, 1H), 7.42 (d, J=9.4 Hz, 1H),6.89 (s, 1H), 5.53 (s, 2H), 4.06 (s, 1H), 3.9, 3.86 (m, 2H), 2.99 (s,6H), 2.71 (s, 4H), 2.38 (s, 3H), 2.25 (s, 3H), 1.71 (s, 4H). (EI-MS):572.0[M+H]⁺.

EXAMPLE 6

Preparation of1-(3-(5-Amino-2-chloro-4-fluoro-3-methylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-N-(1-methylpiperidin-4-group)-1H-1,2,3-triazole-4-carboxamide

Using the methods in application Example 3, replace dimethylaminohydrochloride with 4-amino-1-1methylpiperidin to obtain gray whitesolid. The yield is 49.9%. ¹H NMR (300 MHz, DMSO-d₆) δ9.48 (s, 1H),

9.22 (s, 1H), 8.65 (d, J=2.6 Hz, 1H), 8.52 (d, J=8.2 Hz, 1H), 7.70 (dd,J=8.5, 2.7 Hz, 1H), 7.42 (d, J=8.6 Hz, 1H), 6.89 (d, J=9.2 Hz, 1H), 5.54(s, 2H, 3.78 (s, 1H), 2.92 (t, J=4.6 Hz, 4H), 2.82-2.78 (m, 2H), 2.26(d, J=2.7 Hz, 3H), 2.22 (s, 3H), 2.19 (s, 3H), 2.07-1.86 (m, 4H),1.75-1.66 (m, 4H). (EI-MS): 585.0[M+H]⁺.

EXAMPLE 7

Preparation of 1-(3-(5-amino-2-chloro-4-fluoro-3-ethyl benzoylamino)4-(4-methylpiperazin-1-group)-N-(3-morpholinopropyl)-1H-1,2,3-triazole-4-carboxamide(7)

Using the methods in application Example 3, replace dimethylaminohydrochloride with N-(3-aminopropyl)morpholine, and obtain gray-whitesolid. The yield is 94.2%. ¹H NMR (300 MHz, DMSO-d₆) δ9.51 (s, 1H), 9.22(s, 1H), 8.85 (t, J=5.8 Hz, 1H), 8.66 (s, 1H), 7.71 (dd, J=8.6, 2.7 Hz,1H), 7.43 (d, J=8.8 Hz, 1H), 6.89 (d,

J=9.2 Hz, 1H), 5.53 (s, 2H), 3.62 (t, J=4.6 Hz, 4H), 2.98-2.97 (m, 4H),2.63 (s, 4H), 2.54 (s, 2H), 2.46-2.36 (m, 6H), 2.33 (s, 3H), 2.26 (d,J=2.6 Hz, 3H), 1.74-1.70 (m, 2H). (ESI-MS): 615.1[M+H]⁺. EXAMPLE 8

Preparation of5-amino-2-chloro-4-fluoro-3-ethyl-N-(2-(4-methylpiperazin-1-group)-5-(4-(methylpiperazin-1-Carbonyl)-1H-1,-1H-1,2,3-triazole-1-group)Phenyl)-carboxamide (8)

Using the methods in application Example 3, replace dimethylaminohydrochloride with N-methylpiperazin to obtain gray-white solid. Theyield is 94.2%. ¹H NMR (300 MHz, DMSO-d₆) δ9.51 (s, 1H), 9.22 (s, 1H),8.85 (t, J=5.8 Hz, 1H), 8.66 (s, 1H), 7.71 (dd, J=8.6, 2.7 Hz, 1H), 7.43(d, J=8.8 Hz, 1H), 6.89 (d, J=9.2 Hz, 1H), 5.53 (s, 2H), 3.62 (t, J=4.6Hz, 4H), 2.98-2.97 (m, 4H), 2.63 (s, 4H), 2.54 (s, 2H), 2.46-2.36 (m,6H), 2.33 (s, 3H), 2.26 (d, J=2.6 Hz, 3H), 1.74-1.70 (m, 2H). (ESI-MS):571.0[M+H]⁺.

EXAMPLE 9

Preparation of 1-(3-(5-amino-2-chloro-4-fluoro-3-ethylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-N-(2-Morpholineethyl)-1H-1,2,3-triazole-4-carboxamide (9)

Using the methods in application Example 3, replace dimethylaminohydrochloride with N-Aminoethylmorpholine to obtain gray-white solid.The yield is 72.5%. ¹H NMR (300 MHz, DMSO-d₆) δ9.51 (s, 1H), 8.66 (d,J=2.4 Hz, 1H),

8.56 (t, J=5.8 Hz, 1H), 7.71 (dd, J=8.7, 2.6 Hz, 1H), 7.43 (d, J=8.7 Hz,1H), 6.89 (d, J=9.2 Hz, 1H), 5.54 (s, 2H), 3.58 (t, J=4.6 Hz, 4H), 3.44(s, 2H), 2.96 (t, J=4.8 Hz, 4H), 2.59 (s, 4H), 2.54 (s, 2H), 2.44 (s,4H), 2.30 (s, 3H), 2.26 (d, J=2.6 Hz, 3H). (ESI-MS): 601.0[M+H]⁺.

EXAMPLE 10

Preparation of 1-(3-(5-amino-2-chloro-4-fluoro-3-ethylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-N-(3-aminopropyl)-1H-1,2,3-triazole-4-carboxamide(10)

Using the methods in application Example 3, replace dimethylaminohydrochloride with 1,3-propylene diamine to obtain gray-white solid. Theyield is 64.5%. ¹H NMR (300 MHz, DMSO-d₆) δ8.85 (s, 1H),

8.70 (s, 1H), 8.45 (d, J=2.1 Hz, 1H), 7.36 (dd, J=7.5, 2.0 Hz, 1H), 7.25(s, 1H), 6.84 (d, J=7.5 Hz, 1H), 6.78 (d, J=5.7 Hz, 1H), 4.13 (s, 2H),3.25-3.19 (m, 6H), 2.98 (t, J=4.9 Hz, 4H), 2.67-2.58 (m, 5H), 2.39 (s,3H), 2.19-2.13 (m, 2H), 1.14 (s, 2H). (ESI-MS): 545.0[M+H]⁺.

EXAMPLE 11

Preparation of5-amino-2-chloro-4-fluoro-3-methyl-N-(2-(4-methylpiperazin-1-group)-5-(1H-1,2,3-triazole-1-group)phenyl) benzamide (11)

Using the methods in application Example 1, replace ethyl propiolatewith Trimethyl ethynyl silicon to obtain gray-white solid after threereaction steps. The yield of the three steps is 23.8%. ¹H NMR (300 MHz,DMSO-d₆) δ8.76-8.68 (m, 2H), 8.49 (d, J=2.1 Hz, 1H), 8.18 (d, J=7.5 Hz,1H), 7.27 (dd, J=7.5, 2.0 Hz, 1H), 6.82 (d, J=7.5 Hz, 1H), 6.75 (d,J=5.7 Hz, 1H), 4.15 (s, 2H), 3.20 (t, J=5.1 Hz, 4H), 2.98 (t, J=5.0 Hz,4H), 2.60 (s, 3H), 2.39 (s, 3H). (EI-MS): 444.9[M+Na]⁺.

EXAMPLE 12

Tert-butyl(1-(3-(5-amino-2-chloro-4-fluoro-3-ethylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,2,3-triazole-4-group)aminomethyl ester (12)

Using the methods in application Example 1, replace Methyl propiolatewith tert-butyl ethynyl carbamateto obtain gray-white solid after threereaction steps. The yield of the three steps is 20.1%. ¹H NMR (300 MHz,DMSO-d₆) δ8.70 (s, 1H), 8.59 (d, J=2.0 Hz, 1H), 8.08 (s, 1H), 7.25 (dd,J=7.5, 2.0 Hz, 1H), 7.18 (s, 1H), 6.80 (dd, J=19.7, 6.6 Hz, 2H), 4.17(s, 2H), 3.20 (t, J=5.1 Hz, 4H), 2.98 (t, J=5.1 Hz, 4H), 2.60 (s, 3H),2.39 (s, 3H), 1.50 (s, 9H). (EI-MS): 560.0[M+Na]⁺.

EXAMPLE 13

5-amino-N-(5-(4-amino-1H-1,2,3-triazole-1-group)-2-(4-methylpiperazin-1-group)phenyl)-2-chloro-4-fluoro-3-Methylbenzamide(13)

Dissolve Tert-butyl(1-(3-(5-amino-2-chloro-4-fluoro-3-ethylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,2,3-triazole-4-group)aminomethyl ester (1.0 g, 2.2 mmol) in 20 mL dichloromethane, add 10 mLtrifluoroacetate, stir for 1 h at room temperature, adjust pH=8-9 usingsaturated sodium bicarbonate, extract with dichloromethane, dry theorganic phase with anhydrous sodium sulfate and dry with rotation toobtain grey solie. The yield is 87.3%. ¹H NMR (300 MHz, DMSO-d₆) δ8.70(s, 1H), 8.49 (d, J=2.0 Hz, 1H), 8.08 (s, 1H), 7.25 (dd, J=7.5, 2.0 Hz,1H), 6.80 (d, J=7.5 Hz, 1H), 6.70 (d, J=5.7 Hz, 1H), 5.80 (s, 2H), 4.15(s, 2H), 3.20 (t, J=5.3 Hz, 4H), 2.98 (t, J=5.2 Hz, 4H), 2.60 (s, 3H),2.39 (s, 3H). (EI-MS): 459.9[M+Na]⁺.

EXAMPLE 14

N-(1-(3-(5-amino-2-chloro-4-fluoro-3-ethylbenzoylamino)-4-(4-methylpiperazin-1-group)phenyl)-1H-1,2,3-triazole-4-group)-1-methylpiperidine-4-formamide(14)

Dissolve5-amino-N-(5-(4-amino-1H-1,2,3-triazole-1-group)-2-(4-methylpiperazin-1-group)phenyl)-2-chloro-4-fluoro-3-Methylbenzamide(13) (0.2 g, 0.34 mmol) in 5 mL DMF, add BOP (0.30 g, 0.68 mmol),trimethylamine (0.09 mL, 0.68 mmol) and 1-Methylpiperidine-4-formic acid(97.3 mg, 0.68 mmol), stir for 4 h at room temperature. Dilute thereaction with 50 mL ethyl acetate, remove DMF by washing with saturatedsodium chloride, dry the organic phase with anhydrous sodium sulfate,dry out the organic solvent with rotation to obtain raw product, isolateand purify with chromatography on silica gel column(dichloromethane:methanol=20:1) to obtain grey white solid. The yield is73.9%. ¹H NMR (300 MHz, DMSO-d₆) δ8.72-8.64 (m, 2H), 8.08 (s, 1H), 7.71(s, 1H), 7.25 (dd, J=7.5.20 Hz, 1H), 6.87 (d, J=7.5 Hz, 1H), 6.69 (d,J=5.7 Hz, 1H), 4.16 (s, 2H, 3.20 (t, J=5.3 Hz, 4H), 3.05-2.95 (m, 6H),2.60 (s, 3H), 2.50-2.45 (m, 1H), 2.39-2.37 (m, 6H), 2.14-2.09 (m, 2H),2.05-1.98 (m, 2H), 1.81-1.70 (m, 2H). (EI-MS): 485.1 [M+Na]⁺.

EXAMPLE 15

N-(1-(3-(5-amino-2-chloro-4-fluoro-3-Methylbenzoylamino)-4-(4-methylpiperazingroup)phenyl)-1H-1,2,3-Triazopyridine-4-group) Piperidine-4-formamide(15)

Using the methods in application Example 14, replace1-Methylpiperidine-4-formic acid with 4-piperidinic acid, grey whilesolid is obtained. The yield is 88.7%. ¹H NMR (300 MHz, DMSO-d₆) δ8.70(s, 1H), 8.55 (d, J=2.0 Hz, 1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.26 (dd,J−7.5, 2.0 Hz, 1H), 6.80 (dd, J−23.8, 6.6 Hz, 2H), 4.15 (s, 2H),3.27-3.17 (m, 6H), 2.98 (t, J=5.0 Hz, 4H), 2.82-2.65 (m, 3H), 2.60 (s,3H), 2.39 (s, 3H), 2.03-1.96 (m, 2H), 1.74-1.69 (m, 2H), 1.22 (s, 1H).(EI-MS): 571.1[M+Na]⁺.

EXAMPLE 16

5-amino-N-(5-(4-(4-Aminobutyrylamino)-1H-1,2,3-triazol-1-group)-2-(4-methylpiperazin-1-group)phenyl)-2-chloro-4-fluoro-3-methylbenzoylamino (16)

Using the methods in application Example 14, replace1-Methylpiperidine-4-formic acid with γ-aminobutyric acid to obtain greywhite solid. The yield is 88.7%. ¹H NMR (300 MHz, DMSO-d₆) δ8.70 (s,1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.25 (dd, J=7.5, 2.0 Hz, 1H), 6.80 (dd,J=21.4, 6.6 Hz, 2H), 4.17 (s, 2H), 4.17 (s, 2H), 3.20 (t, J=5.1 Hz, 4H),3.08 3.04 (m, 2H), 2.98 (t, J=5.1 Hz, 4H), 2.60 (s, 3H), 2.50 (t, J=8.2Hz, 2H), 2.39 (s, 3H), 2.10-2.04 (m, 2H), 1.19 (s, 2H). (EI-MS):571.1[M+Na]⁺.

EXAMPLE 17

5-amino-2-chloro-4-fluoro-N-(5-(4-(3-Hydroxypropionylamino)-1H-1,2,3-triazol-1-group)-2-(4-methylpiperazin-1-group)phenyl)-3-methylbenzoylamino

Using the methods in application Example 14, replace1-Methylpiperidine-4-formic acid with 3-Hydroxypropionic acid to obtaingrey white solid. The yield is 84.9%. ¹H NMR (300 MHz, DMSO-d₆) δ8.70(s, 1H), 8.57 (d, J=2.0 Hz, 1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.25 (dd,J−7.5, 2.0 Hz, 1H), 6.80 (dd, J=21.8, 6.6 Hz, 2H), 4.36 (t, J=5.0 Hz,1H), 4.17 (s, 2H), 3.83-3.79 (m, 2H), 3.20 (t, J=5.1 Hz, 4H), 2.98 (t,J=5.0 Hz, 4H), 2.60 (s, 3H), 2.39-2.35 (m, 5H). (EI-MS): 532.1[M+Na]⁺.

EXAMPLE 18

5-amino-2-chloro-N-(5-(4-(4-(dimethylaminomethyl)benzyl)-1H-1,2,3-triazol-1-group)-2-(4-methylpiperazin-1-groupgroup)phenyl)-4-fluoro-3-Methyl benzoyl (18)

Using the methods in application Example 1, replace methyl propiolatewith N,N,-Dimethyl-4-(prop-2-yne-1-group) to obtain grey white solid inthree steps. The yield of the three steps is 34.4%. ¹H NMR (300 MHz,DMSO-d₆) δ8.70 (s, 1H), 8.49-8.44 (m, 2H), 7.74-7.68 (m, 2H), 7.56-7.50(m, 2H), 7.26 (dd, J=7.4, 1.9 Hz, 1H), 6.75 (d, J=5.9 Hz, 1H), 4.16 (s,2H), 3.88 (d, J=1.2 Hz, 2H), 3.20 (t, J=5.1 Hz, 4H), 3.0-2.95 (m, 10H),2.60 (s, 3H), 2.38 (s, 3H). (EI-MS): 606.1[M+Na]⁺.

EXAMPLE 19

5-amino-2-chloro-N-(5-(4-(2-(Dimethylamino)ethyl)-1H-1,2,3-triazol-1-group)-2-(4-methylpiperazin-1-groupgroup)phenyl)-4-fluoro-3-Methyl benzoyl (19)

Using the methods in application Example 1, replace methyl propiolatewith N,N,-dimethyl-but-3-alkyne-1-amine to obtain grey white solid inthree steps. The yield of the three steps is 31.6%. ¹H NMR (300 MHz,DMSO-d₆) δ8.70 (s, 1H), 8.48 (d, J=2.0 Hz, 1H), 8.28 (s, 1H), 7.26 (dd,J=7.5, 2.0 Hz, 1H), 6.82 (d, J=7.5 Hz, 1H), 6.74 (d, J=5.7 Hz, 1H), 4.15(s, 2H), 3.20 (t, J=5.1 Hz, 4H), 2.98 (t, J=5.0 Hz, 4H), 2.71-2.61 (m,4H), 2.60 (s, 3H), 2.40-2.39 (m, 9H). (EI-MS): 606.1[M+Na]⁺.

EXAMPLE 20

5-amino-2-fluoro-N-(3-(4-Ethoxy-1H-1,2,3-triazol-1-group)-2-fluoro-6-(4-methylpiperazin-1-group)phenyl)-4-fluoro-3-Methylbenzoyl (20)

Using the methods in application Example 1, replace4-fluoro-3-Nitroaniline with 2,4-difluoro-3-nitroaniline and methylpropiolate with ethyl ethynyl ether to obtain grey white solid in fivesteps. The yield of the five steps is 12.8%. ¹H NMR (300 MHz, DMSO-d₆)δ8.70 (s, 1H), 7.98 (s, 1H), 7.30 (dd, J=7.5, 5.7 Hz, 1H), 6.74 (d,J=5.7 Hz, 1H), 6.59 (d, J=7.5 Hz, 1H), 4.76-4.72 (m, 2H), 4.15 (s, 2H),3.20 (t, J=5.1 Hz, 4H), 2.98 (t, J=5.0 Hz, 4H), 2.60 (s, 3H), 2.39 (s,3H), 1.56 (t, J=8.0 Hz, 3H). (EI-MS): 507.1 [M+Na]⁺.

EXAMPLE 21

N-(3-(4-((2-amino-2-Oxoethyl)amino)-1H-1,2,3-triazol-1-group)-2-fluoro-6-(4-methylpiperazin-1-group)phenyl)-2-chloro-4-fluoro-5-hydroxyl-3-Methylbenzoyl (21)

Using the method in application Example 1, replace4-fluoro-3-Nitroaniline with 2,4-difluoro-3-nitroaniline, methylpropiolate with 2-(ethynylamino) Acetamide and2-chloro-3-methyl-4-fluoro-5-Nitrobenzoyl chloride with2-chloro-3-methyl-4-fluoro-5-hydroxybenzoyl chloride to obtain whitesolid in five reactions. The yield of the five reactions is 9.7%. ¹H NMR(300 MHz, DMSO-d₆) δ8.70 (s, 1H), 8.07 (d, J=10.4 Hz, 2H), 7.34-7.24 (m,2H), 6.59 (d, J=7.5 Hz, 1H), 5.98 (s, 2H), 5.93 (s, 1H), 4.03 (s, 2H),3.20 (t, J=5.1 Hz, 4H), 2.98 (t, J=5.0 Hz, 4H), 2.60 (s, 3H), 2.38 (s,3H). (EI-MS): 507.1[M+Na]⁺.

EXAMPLE 22

1-(3-(5-amino-2-chloro-4-fluoro-3-Methylbenzoylamino)-2-methyl-4-(4-methylpiperazin-1-group)phenyl)-N-(2-Morpholineethyl)-1H-1,2,3-triazol-4-formamide (22)

Using the methods in application Example 3, replace4-fluoro-3-Nitroaniline with 2-fluoro-4-methyl-3-Nitroaniline andDimethylamino hydrochloride with N-(2-Aminoethyl) Morpholine to obtaingrey white solid. The yield of the six steps is 8.8%.

¹H NMR (300 MHz, DMSO-d₆) δ8.70 (s, 1H), 8.52 (s, 1H), 7.25 (s, 1H),7.13 (d, J=7.5 Hz, 1H), 6.78 (d, J=5.7 Hz, 1H) 6.72 (d, J=7.5 Hz, 1H),4.15 (s, 2H), 3.74 (t, J=4.7 Hz, 4H), 3.55-3.53 (m, 2H), 3.20 (t, J=5.1Hz, 4H), 2.98 (t, J=5.1 Hz, 4H), 2.61-2.59 (m, 5H), 2.51 (t, J=4.7 Hz,4H), 2.40-2.39 (m, 6H). (EI-MS): 615.1[M+Na]⁺.

EXAMPLE 23

N-(3-(4-(3-Aminopropionylamino))-1H-1,2,3-triazol-1-group)-2-methyl-6-(4-methylpiperazin-1-group)phenyl)-2-chloro-4-fluoro-5-hydroxyl-3-Methylbenzoyl (23)

Using the methods in application Example 14, replace4-fluoro-3-Nitrobenzene with 2-fluoro-4-methyl-3-Nitrobenzene,2-chloro-3-methyl-4-fluoro-5-Nitrobenzoyl chloride with2-chloro-3-methyl-4-fluoro-5-Hydroxybenzoyl chloride,1-methylpiperidine-4-carboxylic acid with ß-Alanine to obtain whitesolid in six steps. The yield of the six steps is 7.2%. ¹H NMR (300 MHz,DMSO-d₆) δ8.70 (s, 1H), 8.08 (s, 1H), 7.71 (s, 1H), 7.19 (d, J=7.5 Hz,1H), 7.06 (d, J=5.7 Hz, 1H), 6.72 (d, J=7.5 Hz, 1H), 5.93 (s, 1H), 3.20(t, J=5.1 Hz, 4H), 3.01-2.93 (m, 4H), 2.93-2.91 (m, 1H), 2.60 (s, 3H),2.45 (s, 3H), 2.44-2.42 (m, 2H), 2.38 (s, 3H), 1.24 (s, 2H). (EI-MS):615.1[M+Na]⁺.

EXAMPLE 24

1-(3-(5-amino-2-chloro-4-fluoro-3-Methylbenzoylamino)-4-(4-methyl-1,4-Diazepane-1-group)phenyl)-N-(2-Morpholineethyl)-1H-1,2,3-triazol-4-formamide (24)

Using the methods in application Examples 3, replace N-Methylpiperazinewith N-Methyl homopiperazine and Dimethylamino hydrochloride withN-(2-aminoethyl)morpholine to obtain white solid. The yield of the sixsteps is 7.3%. ¹H NMR (300 MHz, DMSO-d₆) δ8.79 (s, 1H), 8.70 (s, 1H),7.31 (dd, J=7.5, 2.0 Hz, 1H), 7.25 (s, 1H), 6.83 (d, J=7.5 Hz, 1H), 6.70(d, J=5.7 Hz, 1H), 4.16 (s, 2H), 3.74 (t, J=4.7 Hz, 4H), 3.60 (t, J=4.8Hz, 2H), 3.55-3.53 (m, 2H), 3.46-3.44 (m, 2H), 2.91-2.89 (m, 2H),2.61-2.58 (m, 4H), 2.52-2.50 (m, 4H), 2.39 (s, 3H), 2.31 (s, 3H),1.64-1.60 (m, 2H). (EI-MS): 615.1[M+Na]⁺.

EXAMPLE 25

1-3-(5-amino-2-chloro-4-fluoro-3-Methylbenzoylamino)-4-(3,4-Dimethylpiperazine-1-group)phenyl)-N-(2-Morpholineethyl)-1H-1,2,3-triazol-4-formamide (24)

Using the methods in application Example 3, replace N-Methylpiperazinewith 1,2-Dimethylpiperazine and Dimethylamino hydrochloride withN-(2-aminoethyl)morpholine to obtain grey white solid. The yield of thesix steps is 7.3%. ¹H NMR (300 MHz, DMSO-d₆) δ8.80 (s, 1H), 8.70 (s,1H), 8.54 (d, J=2.0 Hz, 1H), 7.31 (dd, J=7.5, 2.0 Hz, 1H), 7.25 (s, 1H),6.81 (d, J=7.5 Hz, 1H), 6.70 (d, J=5.7 Hz, 1H), 4.18 4.04 (m, 7H),3.66-3.53 (m, 2H), 3.35-3.31 (m, 1H), 3.20-3.07 (m, 4H), 2.93-2.87 (m,1H), 2.73 2.58 (m, 2H), 2.51-2.37 (m, 7H), 2.34-2.20 (m, 3H), 1.16 (d,J=6.8 Hz, 3H). (EI MS): 615.1[M+Na]⁺.

1. (canceled)
 2. (canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled) 6.(canceled)
 7. (canceled)
 8. (canceled)
 9. A method of treating acuteleukemia in a subject, the method comprising administering to saidsubject a compound of the formula (I) or a pharmaceutically acceptablesalt thereof:

wherein X is hydrogen, methyl, methoxy or halogen; Y is —CH2-, —O—, —S—,—CO—, —CH2O—, —NR5-, —CONR6- or —NR7CO—, wherein R5, R6, or R7 eachindependently is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, phenyl orsubstituted phenyl, the substituent is halogen, C1-C4 alkyl, C1-C4alkoxy, amino, hydroxyl, thiol, carboxyl, cyano, trifluoromethyl orimidazolyl; M is 0-6; R1 is hydrogen, amino, hydroxyl, thiol, carboxyl,cyano, —CONH2, C1-C4 alkyl, C1-C4 alkoxy, phenyl, substituted phenyl,substituted or unsubstituted nitrogen- or oxygen-containing 3 to 7membered heterocyclic ring, —NR8COR9, —CONR10R11 or —NR10R11, wherein R8is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, phenyl or substituted phenyl,R9 is amino, hydroxyl, C1-C4 alkyl, C1-C4 alkoxy, phenyl or substitutedphenyl, substituted or unsubstituted nitrogen- or oxygen-containing 3 to7 membered heterocyclic ring, R10, R11 independently is hydrogen, C1-C4alkyl, phenyl or substituted phenyl, substituted or unsubstitutednitrogen- or oxygen-containing 3 to 7 membered heterocyclic ring, or R10and R11 are bonded to form nitrogen- or oxygen-containing 3 to 7membered heterocyclic ring, wherein the substituent is halogen, C1-C4alkyl, C1-C4 alkoxy, amino, hydroxyl, thiol, carboxyl, cyano,trifluoromethyl or imidazolyl; R2 is disubstituted or trisubstitutedhalogen, C1-C4 alkyl, C1-C4 alkoxy, trifluoromethyl, nitro or cyano; R3is amino, methylamino, aminomethyl, hydroxyl, hydroxymethyl, thiol or—CONH2; R4 is N-methylpiperazine, 1,2-dimethyl piperazine orN-methylhomopiperazine; and a pharmaceutically acceptable carrier. 10.The method of claim 9, wherein the acute leukemia is an acute leukemiahaving an MLL1 gene rearrangement type.
 11. The method of claim 9, thecompound of the formula (I) or a pharmaceutically is selected from anyof the compounds below: